Congenital heart disease (CHD) is the most common type of birth defect occurring in nearly 1% of live births. Cardiac septation defects account for nearly 50% of CHD. The majority of CHD has a multifactorial origin but increasing evidence suggests that genetic factors play a significant role. The identification of rare cases of familial CHD has been instrumental in uncovering genetic etiologies. The long-term goal of our research is to understand the molecular pathways that underlie the genetic etiologies of human CHD. Utilizing genome-wide linkage analysis, we discovered point mutations in GATA4, which encodes a zinc finger transcription factor critical for normal cardiac development, that were associated with cardiac septal defects and other cardiac malformations. A GATA4 G296S mutation in a large pedigree with CHD revealed a novel functional interaction between GATA4 and TBX5, the genetic cause of Holt-Oram syndrome that is characterized by CHD. Consistent with this in vitro finding, Gata4 and Tbx5 displayed a genetic interaction as mice heterozygous for Gata4 and Tbx5 have a unique cardiac phenotype that results from defective endocardial cushion development. These findings allowed us an entry point to dissect the molecular pathways that are disrupted leading to CHD. While it is clear that Gata4 and Tbx5, as well as other transcription factors, play central roles in cardiac development and CHD, there is a major void in the understanding of the target genes that mediate the actions of these developmental regulators. This is one of the important goals of this proposal. The overall hypothesis is that GATA4 and TBX5 regulate common pathways critical for cardiac septation and other cardiac morphogenetic processes. The aims of this proposal are: Specific Aim 1. To determine the developmental processes and cell lineage(s) in which Gata4-Tbx5 interaction is necessary to result in cardiac defects seen in Gata4 Tbx5 embryos. Specific Aim 2. To understand the role of the Hedgehog signaling pathway in the cardiac phenotype of the Gata4 Tbx5 embryos. Specific Aim 3. To determine the role of the Gata4 G296S mutant protein during embryogenesis. Mice that harbor the G296S mutation in Gata4 will be generated and used to define the molecular and functional in vivo deficits of this point mutation.